Apoptosis-Inducing Activity of Clofazimine in Macrophages

Abstract

Clofazimine is a riminophenazine compound which has been used for the treatment of leprosy since the 1960s. Although the drug is effective in the management of leprosy reactions because of its anti-inflammatory activity, the mechanism leading to the cessation of inflammation is not well understood. In the present study, it was shown that clofazimine exhibits apoptosis-inducing activity in macrophages. When human monocyte-derived macrophages were cultured in vitro in the presence of clofazimine, the cells exhibited a marked decrease in metabolic activity and showed shrinkage in cell size, indicating cell death. Nuclear condensation and fragmentation were also observed by Giemsa and Hoechst 33248 stains. The endonuclease inhibitor ZnCl₂ inhibited the clofazimine-induced cell death. Significant enhancement of caspase-3 activity was observed in clofazimine-treated macrophages and THP-1 cells. Collectively, these results suggest the apoptosis-inducing activity of clofazimine in macrophages, which may also be responsible for the antibacterial properties of clofazimine.

INTRODUCTION

Clofazimine (B663) is a phenazine iminoquinone derivative, specifically, a riminophenazine dye with the empirical formula C₂₇H₂₂C₁₂. In the 1950s, Barry et al. synthesized a large number of compounds by progressive chemical alteration of the anilinoaposafranine molecule, several of which showed antituberculous activity both in vitro and in experimental animals (1). Of these compounds, clofazimine (or Lamprene or B663) was found to be highly active against mycobacteria with the least toxicity. Chang (4) observed the antibacterial activity of clofazimine against Mycobacterium lepraemurium at about the same time as its anti-M. leprae activity was reported by Browne (2) and Browne and Hogenzeil (3). Later, after the introduction of the mouse footpad method of Shepard and Chang (22), its antibacterial activity against M. leprae was demonstrated (18).

Clofazimine has bifunctional activity: antibacterial and anti-inflammatory. It was used in the treatment of leprosy for its antibacterial action against M. leprae. Later, it was also found to possess an anti-inflammatory action which makes it a very useful drug in the treatment of acute reactions, including erythema nodosum leprosum (ENL), neuritis, iritis, etc., although its mechanism of action is unknown (2). In vitro studies on the effect of clofazimine on immune cells have been conducted. Clofazimine increases superoxide anion production and degranulation by stimulated neutrophils. and tumor necrosis factor alpha (TNF-α) potentiates this enhancement (15). The mechanism underlying this pro-oxidative effect seems to involve stimulation of phospholipase A2 (PLA2) activity with subsequent accumulation of arachidonic acid and lysophospholipids, which act as second messengers to activate oxidase (10). In addition, a number of reports have demonstrated the effects of clofazimine that might predict increased immune activity. Lysosomal enzyme activity of cultured macrophages was upregulated by clofazimine (21). Peripheral blood monocytes from healthy volunteers have been demonstrated to exhibit increased major histocompatibility complex class II expression following incubation with clofazimine (25). Increased oxygen uptake during phagocytosis was observed in neutrophils derived from patients with pyoderma gangrenosum during clofazimine therapy (5). Suppressor T-cell activity was decreased in mycobacteria-infected mice during clofazimine treatment (26). However, the mechanisms underlying the anti-inflammatory action of clofazimine are still unclear.

In the present study, we examined the effect of clofazimine on macrophages and found that the drug possessed apoptosis-inducing activity.

MATERIALS AND METHODS

Drug and chemicals.

Clofazimine (Sigma-Aldrich Co., St. Louis, MO), rifampin (catalog no. R3501; Sigma-Aldrich Co.), and dapsone (DDS; Biomol Research Inc., Butler Pike Plymouth Meeting, PA) were dissolved in dimethyl sulfoxide (DMSO) and stored at −30°C until use. Ampicillin was obtained from Sigma-Aldrich Co.

Culture of human macrophages and isolation of bacilli.

Human peripheral blood was obtained under informed consent from healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) gradient centrifugation (12). The cells were suspended in AIM-V medium (Gibco BRL, Invitrogen Corp., Carlsbad, CA), and 1 × 10⁶ PBMCs were cultured in a well of a 24-well tissue culture plate (Falcon; Becton Dickinson Labware, Becton Dickinson and Company, Franklin Lakes, NJ) containing 13-mm round coverslips (Nunc Thermanox coverslips; Nalge Nunc, Thermo Scientific, Rochester, NY) at 37°C in a 5% CO₂ incubator for adherence of monocytes. After 1 h incubation, the coverslips were washed with Hanks' balanced salt solution (HBSS; Sigma-Aldrich Co.) to remove nonadherent cells. The monocytes on the coverslips were cultured in a new 24-well plate containing RPMI 1640 medium (Sigma-Aldrich Co.) supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS; Bio Whittaker Co., Walkersville, MD), 2 mM l-glutamine, and 100 μg/ml ampicillin (RPMI-10F) in the presence of 40 ng/ml of granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN). After 10 days, the cells were differentiated into macrophages and used for experiments. In some experiments, PBMCs were cultured in 35-mm cell culture dishes (Corning Inc., Corning, NY) for adherence, and adherent monocytes were cultured for 10 days. Human monocytic leukemia cell line THP-1 was maintained in RPMI 1640 medium containing 15% fetal bovine serum.

M. leprae (Thai-53 strain) was isolated from the footpads of BALB/c nu/nu mice that had been inoculated with M. leprae 8 months prior to isolation, and the bacillary number was enumerated according to the method of Shepard and Chang (22).

Light and phase-contrast microscopy.

Macrophages on the coverslip were fixed with absolute methanol, followed by performing Giemsa stain (Wako Co., Japan). After they were mounted on a glass slide, the cells were observed under a light microscope (Optiphot-2; Nikon Co., Tokyo, Japan). Photographs were taken with a digital camera (Nikon F70s). Macrophages in 35-mm dishes were incubated in the presence of clofazimine and observed under a phase-contrast microscope (Olympus CKX41 with ×10- and ×20-objective lenses). Photographs were taken with an Olympus DP50 system. Image acquisition and data processing were done using the DP controller software.

Fluorescence microscopy.

Fluorescence staining for DNA was employed. Macrophages were cultured in an 8-well chamber slide (Lab-Tek II chamber slide system; Nalge Nunc). The cells were incubated in the presence of clofazimine and subsequently fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS). Hoechst 33342 dye (Sigma-Aldrich Co.) in PBS was added to the wells at a final concentration of 10 μM, and the slide was incubated for 1 h at 37°C. The cells on the slide were observed under a fluorescence microscope (Olympus BX60 with a ×40-objective lens) equipped with an Olympus DP50 system. The digital images were processed with DP controller software.

Determination of cell death.

Cell viability was determined using the colorimetric method (Cell Titer 96 aqueous nonradioactive cell proliferation assay; Promega Corp., Madison, WI). Briefly, cells in a 24-well plate were incubated in the presence of clofazimine in phenol red-free RPMI 1640 medium containing 10% FBS, followed by addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate solution (formazan reagent). After 1 h incubation at 37°C, an aliquot of medium was transferred into a well of a 96-well plate, and the developed color was measured by a microplate reader at 490 nm. In addition, the activity of lactate dehydrogenase (LDH), released from dead cells into culture supernatants, was measured by a colorimetric assay (Cytotox 96 nonradioactive cytotoxicity assay; Promega Corp.). The color that developed in a sample incubated with LDH substrate was measured by a microplate reader at 490 nm (Vmax; Molecular Devices Corp., Sunnyvale, CA).

DNA electrophoresis.

THP-1 cells or macrophages were harvested from the culture, and DNA was purified by a spin column method (E.Z.N.A. tissue DNA kits; Omega Bio-Tek, Norcross, GA). Briefly, 5 × 10⁶ cells incubated in the presence of clofazimine were harvested, centrifuged at 2,000 rpm for 5 min, washed once with PBS, and resuspended in PBS. Protease was added, the mixture was heated at 65°C for 5 min, and buffer BL was added. After the mixture was heated at 70°C for 10 min, ethanol was further added. The mixture was applied to a HiBind spin column and centrifuged. DNA bound to the column was finally eluted, and the DNA preparation was subjected to electrophoresis in a 1% agarose gel, followed by ethidium bromide staining, and DNA was visualized by UV transillumination.

Western blotting.

THP-1 cells or macrophages incubated with clofazimine were washed once with PBS(−) and lysed in lysis buffer (CelLytic-M; Sigma-Aldrich Co.) containing 2 protease inhibitor cocktails (phosphatase inhibitor cocktail 1 and phosphatase inhibitor cocktail 2; Sigma-Aldrich Co.). In the case of clofazimine-treated adherent macrophages, the cells were scraped off the dishes with a rubber policeman. The lysates were incubated for 10 min on ice and centrifuged at 13,000 rpm for 5 min. The protein concentration was determined. Ten micrograms of total protein was loaded onto an SDS-PAGE gel. After running the electrophoresis, the proteins in the gel were transferred onto an Immobilon PSQ membrane (Millipore Corporation, Billerica, MA). After washing with Tris-buffered saline (2.42 g Tris base and 8 g NaCl per 1 liter, pH 7.6) containing 0.05% Tween 20 (TBS-T), the membrane was blocked with 5% skim milk (Amersham ECL Plus Western blotting reagent pack; GE Healthcare Life Sciences, Amersham Place, Buckinghamshire, United Kingdom) for 1 h at room temperature. The membrane was washed 3 times with TBS-T and incubated overnight with 1:3,000-diluted primary anticleaved caspase-3 antibody (cleaved caspase antibody sampler kit; Cell Signaling Technology Inc., Danvers, MA). The membrane was then incubated with 1:10,000-diluted horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, proteins were detected by incubating the membrane with HRP substrate (Immobilon Western chemiluminescent HRP substrate; Millipore Corporation), and the membrane was exposed to X-ray film (Amersham Hyperfilm ECL; GE Healthcare). For reprobing of the membrane, the membrane was washed with TBS-T and incubated with stripping buffer (Restore Plus Western blot stripping buffer; Pierce, IL). After the membrane was blocked, it was used again for probing different antibodies, such as cleaved caspase-9 and poly(ADP-ribose) polymerase (PARP) antibodies (cleaved caspase antibody sampler kit; Cell Signaling Technology Inc.) and beta-actin antibody (Cell Signaling Technology Inc.).

Colorimetric caspase assay.

Colorimetric substrates for caspases were used to determine caspase-3 activity (colorimetric caspase assay kits; Biovision Research Products, CA) in lysates of cells incubated in the presence of clofazimine. Briefly, 5 × 10⁶ cells were pelleted and lysed with chilled lysis buffer. After centrifugation, the supernatant was transferred to a new tube, and reaction buffer and a substrate for caspase-3, Asp-Glu-Val-Asp–p-nitroanilide, were added to the tube. After incubation for 2 h at 37°C, the samples were transferred into a well of a 384-well plate and read by a plate reader at 405 nm (Infinite F200; Tecan Systems Inc., San Jose, CA). The background reading was obtained by subtracting the reading for the reaction buffer from the reading for the lysate samples.

PGE₂ assay.

The amount of prostaglandin E₂ (PGE₂) in the culture supernatants was measured by enzyme-linked immunosorbent assay (catalog no. 514010; Cayman Chemical Co., MI).

RESULTS

Morphological changes observed after treatment with clofazimine.

Macrophages differentiated from human monocytes were incubated in the presence of 10 μg/ml of clofazimine for 20 h. The change in cell morphology was observed under a phase-contrast microscope. As shown in Fig. 1B, in the presence of clofazimine, the cells exhibited shrinkage in cell size and membrane blebbing. The death of more than 80% of cells was observed (Fig. 1B). As a control, Fig. 1A shows the normal morphology of macrophages. By Giemsa stain, too, these clofazimine-treated cells exhibited shrinkage in cell size, accompanied by the appearance of fragmented smaller nuclei (arrow in Fig. 1D), suggesting the apoptotic nature of the cells. Nontreated macrophages showed intact nuclei (arrow in Fig. 1C). Again, the change of nuclear structure was confirmed by Hoechst dye staining. Under a fluorescence microscope, nuclear condensation and membrane blebbing were observed in the clofazimine-treated cells (Fig. 1F and G), in contrast to normal cells, which showed intact nuclei (Fig. 1E). Similar fragmentation or condensation of chromatin was observed in THP-1 cells (data not shown). Such morphological changes were not observed in THP-1 cells treated with rifampin or dapsone at a concentration up to 50 μg/ml. Also, DMSO, which was used as a solvent for clofazimine at a concentration of 0.2%, had no effect on cell morphology or cell functions (negative control).

Fig. 1.

Cell death induced in macrophages by clofazimine. Photographs were taken under a phase-contrast microscope (A and B) with a ×20-objective lens. (B) Human monocyte-derived macrophages were incubated in the presence of 10 μg/ml of clofazimine for 20 h. (A) Cells cultured in the absence of clofazimine showed normal morphology. Giemsa stain of clofazimine-treated macrophages was also performed (C and D). Human monocyte-derived macrophages were incubated in the presence of 10 μg/ml of clofazimine (D) or in the absence of clofazimine (C) for 24 h. Photographs were taken under a light microscope with a ×100-objective lens. Fragmentation of the nucleus was significant in the clofazimine-treated cells (arrow in panel D), in contrast to the intact morphology of the nucleus in normal cells (arrow in panel C). Nuclear condensation and fragmentation of clofazimine-treated macrophages were also confirmed under a fluorescence microscope (E to G). Macrophages were incubated in the presence of 10 μg/ml of clofazimine, followed by fixation and stained with a nucleus-staining dye, Hoechst 33342. The cells were observed under a fluorescence microscope (×40-objective lens). Cells cultured without clofazimine (E), clofazimine-treated cells (F and G), and a phase-contrast image of panel G (H) are shown.

Cell death-inducing activity of clofazimine determined by colorimetric assay.

Cell death was determined by a biochemical analysis using a colorimetric method. The conversion of the tetrazolium compound into soluble formazan is accomplished by metabolically active cells. When higher concentrations up to 10 μg/ml of clofazimine were employed in macrophage cultures, decreased color intensity of soluble formazan was observed, indicating cell death (Fig. 2A). Cell death was also observed in THP-1 cells (Fig. 2B). Hansen's disease is caused by infection of macrophages with M. leprae; therefore, we are curious to know whether M. leprae infection affects the cell death-inducing activity of clofazimine. When we infected macrophages with M. leprae at a multiplicity of infection (MOI) of 10 or 30, we found no significant difference in the induction of cell death in the presence of 10 μg/ml clofazimine, indicating that the bacilli did not inhibit or enhance clofazimine-induced cell death (Fig. 2C). Another method of determining cell death is by measurement of LDH release from dead cells. As shown in Fig. 3, more LDH release was observed in the manner dependent on the concentration of clofazimine.

Fig. 2.

Clofazimine-induced cell death in macrophages and THP-1 cells. Human monocyte-derived macrophages (A) and THP-1 cells (B) were incubated with various concentrations of clofazimine for 24 h, followed by determination of viable cells by the Cell Titer 96 cell proliferation assay. The results are representative of three independent cell culture tests. The cell death-inducing effect of clofazimine in the presence of M. leprae was also examined. Monocyte-derived macrophages were infected with M. leprae at an MOI of 10 or 30 per cell for 24 h. The infected cells were further incubated with 10 μg/ml clofazimine for another 24 h, followed by determination of viable cells by Cell Titer 96 cell proliferation assay (C). The results are representative of three independent cell culture tests. OD, optical density.

Fig. 3.

LDH release from clofazimine-treated macrophages. Human monocyte-derived macrophages were incubated in the presence of the indicated concentrations of clofazimine for 24 h, and the LDH activity was measured. The results were obtained from triplicate cultures and are representative of three independent cell culture tests. OD, optical density.

Clofazimine treatment induces DNA ladder formation in macrophages.

We examined the condition of DNA in clofazimine-treated THP-1 cells. Agarose gel electrophoresis showed fragmentation of DNA into integer multiples of 180 bp, a so-called DNA ladder (Fig. 4A), suggesting that DNA endonuclease was activated by clofazimine treatment. Therefore, we examined the effect of one of the apoptosis inhibitors, ZnCl₂, which is known to possess suppressing activity for endonuclease, and found that clofazimine-induced DNA fragmentation in THP-1 cells was completely blocked by ZnCl₂ treatment even at a low concentration of 0.25 mM ZnCl₂ (Fig. 4B), although it is still not clear whether ZnCl₂ can directly block the activity of clofazimine. Moreover, it was evident that neither cell death nor DNA fragmentation was induced by other antileprosy drugs, such as DDS or rifampin (Fig. 4C).

Fig. 4.

DNA ladder formation in clofazimine-treated THP-1 cells and effects of other antileprosy drugs on DNA ladder formation. (A) THP-1 cells were incubated in the presence of the indicated concentrations of clofazimine for 4 h, followed by purification of DNA for agarose gel electrophoresis. An ethidium bromide-stained agarose gel is shown. (B) An endonuclease inhibitor, ZnCl₂, was examined for its effect on clofazimine-induced ladder formation. THP-1 cells were incubated in the presence of 10 μg/ml clofazimine and ZnCl₂ for 4 h. DNA was purified for electrophoresis. (C) THP-1 cells were incubated in the presence of clofazimine, DDS, and rifampin for 4 h, followed by purification of DNA. An ethidium bromide-stained agarose gel is shown. Lanes M, molecular weight marker.

Clofazimine-induced cell death is mediated by activation of caspase-3.

Caspases are known to be central regulators of apoptotic cell death, and caspase-3, which locates downstream of the caspase pathway, is one of the key executioners of apoptosis. Upon apoptotic stimulation, caspases are cleaved into active fragments. Figure 5 shows a Western blot analysis of extracts from THP-1 cells and macrophages cultured in the presence of clofazimine. Enhanced expression of cleaved caspase-3 was detected in cells (Fig. 5A and B). In addition, caspase-9 was also cleaved. A DNA-repairing enzyme, PARP, which is cleaved by caspase-3, was significantly activated in clofazimine-treated THP-1 cells (Fig. 5A). We next measured the caspase activity by colorimetric assay (Fig. 5C). The induction of caspase-3 by clofazimine was significantly high in macrophages as well as THP-1 cells.

Fig. 5.

Expression of caspase in clofazimine-treated THP-1 cells and macrophages. THP-1 cells were incubated in the presence of the indicated concentrations of clofazimine for 6 h, and cell lysates were processed for detection of cleaved caspase-3, caspase-9, and PARP by Western blotting (A). Similarly, monocyte-derived macrophages were incubated in the presence of 10 μg/ml of clofazimine, and the cell lysates were examined for cleaved caspase-3 and caspase-9 expression (B). The caspase activity in clofazimine-treated macrophages and THP-1 cells was analyzed. Macrophages were incubated in the presence of 10 μg/ml of clofazimine for 6 h, and the caspase-3 activity in the cell lysates was determined by colorimetric assay (C). The results are representative of three independent cell culture tests.

Clofazimine enhanced PGE₂ production in M. leprae-infected macrophages.

Monocyte-derived macrophages were preincubated in the presence of clofazimine for 4 h, followed by replenishment with M. leprae-containing medium for 20 h. The culture supernatants were collected, and the PGE₂ concentration was measured. As shown in Fig. 6, clofazimine clearly enhanced PGE₂ production in macrophages.

Fig. 6.

Enhancement of PGE₂ production by clofazimine in M. leprae-infected macrophages. Macrophages were preincubated in the presence of the indicated doses of clofazimine for 4 h, followed by infection with M. leprae (5 × 10⁶/well), and the culture was continued for another 20 h. The amount of PGE₂ in the culture supernatants was measured. The results are representative of three independent cell culture tests.

DISCUSSION

Riminophenazines are structurally phenazine compounds which were derived from lichens historically and were targeted for treatment of tuberculosis. The first clinically developed phenazine compound was clofazimine, whose activity has been extended to other mycobacterial diseases (1, 17). In test animals, the drug was found to inhibit the growth of mycobacteria in vivo, as well as in vitro (22), but the molecular mechanism of clofazimine in inducing anti-M. leprae activity is still not yet clear.

In the present study, it was found that both human monocyte-derived macrophages and THP-1 cells exhibited marked decreases in their metabolic activity in the presence of 10 μg/ml clofazimine. Under a phase-contrast microscope, 80% of the cells showed irregular morphology with shrinkage in cell size, and by a precise time course study, it was revealed that the morphological changes were evident from 6 h incubation with clofazimine. From this early time point, the cell body began to shrink, accompanied by membrane blebbing, which was also evident from Giemsa stain and Hoechst staining of the nuclei (Fig. 1). Interestingly, the dose of clofazimine (10 μg/ml) required to cause cell death was equivalent to the dose required to exhibit anti-M. leprae activity in vitro by radiorespirometry (data not shown), the dose of which is in concordance with the dose required to kill M. leprae reported by Franzblau and O'Sullivan (7). Moreover, in our study, at 5-μg/ml concentrations of clofazimine, M. leprae viability was lowered in in vitro experiments with M. leprae-infected macrophages, and with this dose, M. leprae was found not to inhibit clofazimine-induced cell death. Therefore, clofazimine might inhibit mycobacterial growth through an alternative way by inducing apoptosis of host cells. Although the concentration of clofazimine in sera of patients taking regular doses of the drug is as low as 1 to 2 μg/ml, fat-soluble clofazimine readily accumulates in cells. In one patient, 7 months treatment with clofazimine (200 mg/day) resulted in accumulation of needle-shaped crystal inclusions in his alveolar macrophages (20). In another report, clofazimine-induced crystal-storing histiocytosis was observed in a leprosy patient (23). So, we are of the opinion that in some cells, the concentration of clofazimine is higher (10 to 20 μg/ml) than in others, so we have used a concentration of 10 μg/ml for our experiments.

Normally, cells undergo distinct morphological changes when they progress through either necrosis or apoptosis. Necrosis occurs when cells are exposed to an extreme variance from physiological conditions, resulting in damage to the plasma membrane. As such, necrosis is characterized by cell swelling and disruption of cellular organelles, with little change in the chromatin initially. In contrast, apoptotic cells shrink in size, undergo membrane blebbing, and exhibit marked alterations in their chromatin structure at an early stage under normal physiological conditions. As mentioned earlier, treatment with clofazimine resulted in highly condensed chromatin within the nucleus and membrane blebbing, indicating macrophages undergoing apoptosis. To confirm this, DNA from clofazimine-treated THP-1 cells was examined. Fragmented DNA was demonstrated, suggesting that DNA endonuclease was activated causing apoptosis.

We observed that M. leprae by itself does not induce apoptosis of human cells. Similarly, infection of mouse macrophages with viable M. leprae was shown not to induce apoptosis (11). Although apoptosis is induced when macrophages infected with M. leprae are treated with clofazimine, the host cell viability does not change significantly in the presence of M. leprae. Nevertheless, the viability of M. leprae in macrophages was significantly lower in clofazimine-treated cells than infected cells not treated with clofazimine (data not shown). Therefore, we can speculate that clofazimine induces apoptosis of M. leprae-infected macrophages, which in turn inhibits M. leprae growth.

Consequently, we investigated the pathways involved in the execution of macrophage apoptosis (6, 14). We observed enhanced expression of cleaved caspase-3, caspase-9, and PARP following clofazimine treatment in THP-1 cells (Fig. 5A). Colorimetric assay also indicated enhanced caspase-3 activity in both macrophages and THP-1 cells treated with clofazimine (Fig. 5C), suggesting the involvement of caspases in clofazimine-induced apoptosis.

Apoptosis has been shown to be effective in therapy of chronic inflammatory diseases (16). An immunomodulatory drug, thalidomide, is used for treatment of ENL in leprosy patients, and its anti-inflammatory activity is believed to be through the downregulation of production of the proinflammatory cytokine TNF-α (19). Gockel et al. showed that thalidomide induces apoptosis in human monocytes (8). Clofazimine is known to have a favorable influence on the reversal reaction in human leprosy (2). Browne and Hogenzeil found that clofazimine controlled persistent exacerbations in patients who were corticosteroid dependent for controlling the passing reactions, including ENL, and suggested that the drug may exert a suppressive effect on the development of acute exacerbation in lepromatous leprosy (3). These observations have been followed by those of later workers, and one of the special indications for use of clofazimine is the presence of acute reactions or a chronic recurrent reaction in lepromatous leprosy. These clinical data suggest that the mechanisms underlying the action of the drug in these leprosy patients mainly seem to be anti-inflammatory, although there is no direct evidence. Macrophages are capable of elaborating a series of biochemical products with potent immunomodulatory activities. We have observed enhancement of the production of PGE₂, when macrophages were pretreated with clofazimine. PGE₂ is released from arachidonic acid by PLA2. The enzyme is reported to be stimulated in clofazimine-treated neutrophils (10). With respect to B-cell function, prostaglandins of the E series (PGE) inhibit both B-cell proliferation and the generation of antibody-forming cells, and also, B-cell tolerance is induced by PGE₂ (9, 24). T-cell proliferation is also suppressed by PGE₂ (13). The reaction to leprosy involves antibody (ENL caused by immune complex) and cells (delayed-type hypersensitivity mediated mainly by T cells). The mechanisms underlying the immunomodulatory role of clofazimine are still not clear, but the present study clarifies certain aspects. Apoptosis induced in macrophages might explain the anti-inflammatory activities of clofazimine in vivo.

In conclusion, our findings suggest that clofazimine induced apoptosis of macrophages through the activation of caspases. The data indicate that the action of clofazimine in leprosy patients may be at least partially mediated by apoptosis.